Plasminogen is a zymogen of plasmin as shown in FIG. 1A. The amino acid sequence of the human plasminogen precursor is depicted in FIG. 1B. Human plasminogen contains 791 amino acids (precursor=810 amino acids) with a molecular weight of about 90 kDa and a pl of approximately 7.0, although differential glycosylation and/or removal of the N-terminal activation peptide can result in a pl range of 6.2 to 8.0. It is a single-chain protein with 24 intra-chain disulfide bridges, 5 kringle domains (involved in the binding to fibrin and to the inhibitor α2-antiplasmin), a serine protease domain (P), and an activation peptide (AP) consisting of the first 77 amino acids. There is one N-linked glycosylation site and one O-linked site, although a second O-linked site has been identified (Goldberg, 2006). Approximately 70% of the Plasminogen in circulation contains only O-linked glycosylation while the rest contains both N- and O-linked sugars.
Native Plasminogen is produced in two main forms, Glu-Plasminogen (Glu-Pg) and Lys-Plasminogen (Lys-Pg), named for the N-terminal amino acid of either glutamic acid or lysine. Glu-Pg is composed of the entire amino acid sequence designated by the gene sequence (excluding the activation peptide), while Lys-Pg is the result of a cleavage of the Glu-Pg between Lys-77 and Lys-78 (underlined in FIG. 1B). The circulating half-life of Lys-Pg is considerably shorter than Glu-Pg (2-2.5 days for Glu-Pg, 0.8 days for Lys-Pg). Glu-Pg is the dominant form of Pg present in plasma with very little Lys-Pg detected in the circulation (Violand, B. N., Byrne, R., Castellino F. J. (1978) The effect of α-,ω-Amino Acids on Human Plasminogen Structure and Activation. J Biol Chem. 253 (15): 5395-5401; Collen D, Ong E B, Johnson A J. (1975) Human Plasminogen: In Vitro and In Vivo Evidence for the Biological Integrity of NH2-Terminal Glutamic Acid Plasminogen. Thrombosis Research. 7 (4):515-529).
Plasminogen is synthesized in the liver and secreted into plasma. Plasminogen is distributed throughout the body and when conditions are present for activation, the plasminogen pro-enzyme is converted to the active enzyme, plasmin, by tissue-type plasminogen activator (t-PA) or by urokinase plasminogen activator (u-PA). Plasmin then degrades fibrin and converts latent matrix metalloproteinases (pro-MMPs) into active MMPs, which in turn further degrade extracellular matrix (ECM) as part of the tissue healing/remodeling process. Plasminogen activation mediated by t-PA is primarily involved in fibrin homeostasis, while plasmin generation via u-PA, forming a complex with its receptor u-PAR, plays a role in tissue remodeling.
Plasmin is or was investigated for its potential use for the clearance of thrombotic occlusions in artificial devices and hemodialysis grafts, and for the treatment of posterior vitreous detachment (PVD) (U.S. Pat. No. 6,969,515; US 2010/0104551).
Plasminogen is investigated for its use in therapeutic indications such as wound healing, healing of a tympanic membrane perforation, healing of periodontal wound, infectious disease, oral health, diabetic ulcer, thrombolysis indications, such as coronary thrombosis, reperfusion injury to tissue, ischemia, infarction, brain edema, improvement of microcirculation, and modulation of complement pathway (U.S. Pat. Nos. 8,637,010; 8,679,482; 8,318,661; WO 95/12407; EP 0,631,786). As yet, no plasminogen is currently on the pharmaceutical market as a drug.
Historically, Lys-Pg was pharmaceutically commercialized for a period of time for hematologic purposes and has not been scientifically or medically used since 2000. A formulation of Lys-Pg is described in Schott et al., 1998, The New England Journal of Medicine, Vol. 339, No. 23, pp. 1679-1686). While available Lys-Pg was clinically used and investigated for the treatment of ligneous conjunctivitis, a clinical manifestation of the underlying condition hypoplasminogenimia (plasminogen deficiency type I). Hence systemic administration of Lys-Pg concentrates has been tested. Kraft et al. (Kraft J, Lieb W, Zeitler P, Schuster V. (2000) Ligneous conjunctivitis in a girl with severe type I plasminogen deficiency. Graefes Arch Clin Exp Ophthalmol. 238(9):797-800) reported that daily infusion of Lys-Pg in a child with severe hypoplasminogenemia resulted in partial resolution of the conjunctival pseudo membranes. Schott et al. (1998) reported that, in a 6-month-old child, treatment with Lys-Pg preparation as a continuous infusion and later as daily bolus injections led to complete regression of the ligneous conjunctivitis within 4 weeks and normalized hyperviscous secretions in the respiratory tract as well as skin wound healing. Schuster et al (Schuster V, Hugle B, Tefs K (2007) Plasminogen deficiency. J Thromb Haemost 5(12):2315-2322) reported that plasminogen levels in patients with homozygous or compound heterozygous (the presence of two different mutant alleles at a particular gene locus, one on each chromosome of a pair) hypoplasminogenemia ranged from <1 to 9 mg/dL for plasminogen antigen plasma levels and <1% to 51% for functional plasminogen activity. It is important to note that the majority of these patients have some residual plasminogen activity levels. Thus, plasminogen replacement is expected to be effective, as it is an endogenous protein and is not expected to have immunogenicity or fibrinolytic activity concerns. Although systemic or topical plasminogen concentrates have been clearly documented as effective therapy leading to resolution and halts re-formation of the lesions (Watts P, Suresh P, Mezer E, Ells A, Albisetti M, Bajzar L, Marzinotto V, Andrew M, Massicotle P, Rootman D (2002) Effective treatment of ligneous conjunctivitis with topical plasminogen. Am J Ophthalmol 133(4):451-455, Heidemann D G, Williams G A, Hartzer M, Ohanian A, Citron M E (2003) Treatment of ligneous conjunctivitis with topical plasmin and topical plasminogen. Cornea 22(8):760-762; and Schott, 1998), no purified plasminogen product for topical or for systemic therapy is commercially available.
Only very recently, clinical trials were undertaken to utilize Glu-Pg for the treatment of the type I plasminogen deficiency (Clinical Trials.gov Identifier: NCT02312180) and one of its clinical manifestation ligneous conjunctivitis utilizing a localized eye drop of Glu-Pg (ClinicalTrials.gov Identifier: NCT01554956).
Proteins may be stabilized either by changing their structural characteristics (internally) and/or by controlling the components in contact with them (externally). Some proteins have raised particular challenges with respect to handling and behaviour in pharmaceutical formulations due to their physico-chemical characteristics which often unfortunately leads to structural instability. They can actually undergo various types of degradations as exemplified by the following: 1) chemical processes leading to the formation of related impurities, which may involve hydrolysis, oxidation reactions, deamidation or structural rearrangements such as iso-asp or intramolecular truncations or 2) a physical process giving rise to aggregation/polymerization thus generating structural alterations which may impact on the biological activity and potentially enhance immunogenicity.
Formulation development generally refers to a process in which an active pharmaceutical ingredient (API) is characterized to a sufficient extent that it can be converted to a pharmaceutically acceptable drug substance. Biophysical characterization of drug substances must be performed to confirm that the correctly folded and biologically active structure is present. Several spectroscopic techniques (Fluorescence, CD, DSC, DLS, etc.) can be used to examine the tertiary structure of proteins in solution and to assess the stability and effect of different formulations conditions on protein structure. (Volkin, D. B. et al. “Preformulation studies as an essential guide to formulation development and manufacture of protein pharmaceutical.” Development and manufacture of protein pharmaceuticals. Edited by Steve L. Nail and Michael J. Akers, Kluwer Academic 2002 Chapter 1, page 1-39; Cheng, W. et al. “Comparison of High-Throughput Biophysical Methods to Identify Stabilizing Excipients for a Model IgG2 Monoclonal Antibody: Conformational Stability and Kinetic Aggregation Measurements” Journal of Pharmaceutical Sciences, Vol. 101, No 5, page 1701-1720, 2012). In addition, often the pharmaceutical industry confirms the structural integrity of the API by performing a biologically assay for the release of the drug substance. Taken together they confirm that the preferred formulation has not inadvertently been modified by the formulation. Plasminogen is a protein that is mainly used in the preparation of plasmin for thrombolysis indications, such as coronary thrombosis, clearance of thrombotic occlusions in artificial devices and hemodialysis grafts, and for reperfusion injury to tissue, treating ischemia, infraction, brain edema, or for improving the microcirculation (WO 95/12407; U.S. Pat. No. 6,969,515; EP 0,631,786).
The administration of plasminogen has also been found useful in many therapeutic indications such as thrombolysis indications, such as coronary thrombosis, treating reperfusion injury to tissue, treating ischemia, infarction, brain edema, or for improving the microcirculation, wound healing, healing of a tympanic membrane perforation, healing of periodontal wound, infectious disease, oral health, diabetic ulcer, plasminogen-deficient subjects, and modulation of complement pathway (WO 95/12407; EP 0,631,786 U.S. Pat. No. 8,637,010; 8,679,482; 8,318,661).
There are several challenges encountered when formulating plasminogen. Some problems result from the plasmin contamination of the plasminogen formulation, which degrades plasminogen. Various approaches have been used for avoiding degradation of plasminogen including the addition of aprotinin, lysine, phenylmethanesulphonyl fluoride, soybean trypsin inhibitor or serine protease inhibitor (U.S. Pat. No. 4,177,262; 4,361,653, 5,304,383).
Other difficulties are represented by the turbidity or the presence of filamentous substances when plasminogen is solubilised in an aqueous solvent. To overcome this drawback, it has been proposed to combine plasminogen for example with a nonionic surfactant or with a mixture of sucrose, amino acid and albumin (WO 94/15631).
There is no known commercial formulation of Glu-Pg for human therapeutic use. The only formulations of Glu-Pg that are known are for research only.
There is a need for the development of a pharmaceutical composition of plasminogen.
The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety.